Borrelia burgdorferi, a spirochete, is the causative agent of Lyme disease. It is a vector borne pathogen, transmitted by ticks of the Ixodes scapularis and Ixodes pacificus complexes in the United States. Burgdorfer W., B. burgdorferi: Its Relationships to Tick Vectors, In Staken (ed): Lyme Borreliosis, II, Zbl Bakt Supplement 18, Stuttgart-New York, Gustav Fischer, 8-13, 1989. Infection with B. burgdorferi is the most common tick-borne infectious disease in the United States. In endemic areas, between 30 and 90% of Ixodes ticks are infected with this organism.
The illness caused by B. burgdorferi is a multisystem infection that affects the skin, central nervous system, peripheral nerves, the heart and the joints, with nerve and joint involvement being the most common. Steere, A. C., S. E. Malawista, J. A. Hardin, S. Ruddy, W. Askenase, and W. A. Andiman, Erythema Chronicum Migrans and Lyme Arthritis: the Enlarging Spectrum, Ann Intern Med. 86:685-698, 1977a. The dissemination of spirochetes over time from the site of infection has been documented in a mouse model. Barthold, S. W., D. H. Persing, A. L. Armstrong, and R. A. Peeples, Kinetics of B. burgdorferi Dissemination and Evolution of Disease after Intradermal Inoculation of Mice, AM J. Pathol, 139:263-273, 1991. In these experiments it was shown that the spirochetes were multifocal in distribution with a predilection for collagenous connective tissue of joints, heart, arteries, nerves, muscles, skin and other tissues. In humans, nerve, joint and heart involvement is usually manifested when the disease reaches a chronic state. The pathogensis to a chronic state of nerve involvement, termed neuroborreliosis, is unclear, but may be due to a direct effect of the spirochetes at the site of infection, the host response to B. burgdorferi, or the host response to tissue antigens that may mimic those of Borrelia burgdorferi. Fikrig, E., R. Berland, M. Chen, S. Williams, L. H. Signal, and R. A. Flavell, Serological Response to B. burgdorferi Flagellin Demonstrates an Epitope Common to a Neuroblastoma Cell Line, Proc. Natl. Acad. Sci. 90:183-187, 1993. For example, spirochetes can be detected in the cerebrospinal fluid of patients with neuroborreliosis with a corresponding high level of antigen specific T-cells which suggests that a local immune response may be involved in the disease. However, the presence of spirochetes has yet to be demonstrated in biopsy specimens of affected nerve tissue suggesting that clinical manifestations may be due to some type of molecular mimicry. Indeed, it has been shown that antibodies reactive to a certain epitope of the B. burgdorferi flagellin molecule cross react with a neuroblastoma cell line. Fikrig, E., R. Berland, , M. Chen, S. Williams, L. H. Sigal , and R. A. Flavell, Serological Response to B. burgdorferi Flagellin Demonstrates an Epitope Common to a Neuroblastoma Cell Line, Proc. Natl. Acad. Sci. 90:183-187, 1993. Development of arthritic symptoms is similarly complex. Patients who develop arthritis usually show involvement of the knee joint with an increase of polymorphonuclear granulocytes in the synovial fluid. Steere, A. C., S. E. Malawista, and D. R. Snydman, Lyme Arthritis: An Epidemic of oligoarticular Arthritis in Children and Adults in Three Connecticut Communities, Arthritis Rheum. 20:7-17, 1977. It has been hypothesized that spirochetes trigger a local immune response with autoreactive features in the joint, symptoms which may continue after the organisms no larger are viable. Girouard, L., D. C. Laux, S. Jindal, and D. R. Nelson, Immune Recognition of Human Hsp60 by Lyme Disease Patient Sra, Microb. Pathog. 14:287-297, 1993; and Steere, A. C., J. Feld, and R. Winchester, Association of Chronic Lyme Arthritis with Increased Frequencies of DR4 and 3, Arthritis Rheum. 31:98 (Abstract), 1988. As with nerve biopsies, culturing of spirochetes from synovial fluid of the joints is exceedingly difficult, being reported only twice, and direct observation is also rare. These phenomena are especially true as the disease progresses. Recent advances in the ability to detect small amounts of DNA using polymerase chain reaction (PCR) has led to conflicting results regarding the presence of spirochetes in the joint. Nocton, J. J., F. Dressler, B. J. Rutledge, P. N. Rys, D. H. Persing, and A. C. Steere, Detection of B. burgdorferi DNA by Polymerase Chain Reaction in Synovial Fluid in Lyme Arthritis, N. Engl. J. Med. (In press); and Malawista, S. E., T. L. Moore, D. E. Dodge, T. J. White, R. T. Schoen, and D. H. Persing, Failure of Multitarget Detection of Borrelia burgdorferi-Associated DNA Sequences in Synovial Fluids of Patients with Juvenile Rheumatoid Arthritis: A Cautionary Note, Arthritis Rheum. 35:246-247, 1992. Thus, as with neuroborreliosis, there appear to be two possibilities: septic arthritis with live organisms in the joint, and reactive arthritis where a microbial antigen at a remote site stimulates an immune response involving either antibodies or cytotoxic mononuclear cells that cross-react with a compartment of host tissue.
The humoral response to B. burgdorferi has been well documented. There is a potent response to the 41 kDa flagellar protein early during the course of infection followed by an antibody response to various other proteins, notably Hsp60, Hsp70, a 66 kDa protein and other lower molecular weight proteins. Chronic infection generally results in development of an antibody response to the outer surface lipoproteins A and B (OspA and OspB). Barbour, A. G., W. Burgdorfer, E. Grunwaldt, and A. C. Steere, Antibodies of Patient with Lyme Disease to Components of the Ixodes Dammini Spirochete, J. Clin. Invest. 72:504-510, 1983. These and other outer surface proteins have been given a great deal of attention because of their potential as vaccine candidates and because of their possible virulence functions. Antibodies against OspA have been shown to be protective against Lyme disease--mice immunized with recombinant OspA are resistant to infection with Borrelia burgdorferi. Fikrig, E., S. W. Barthold, F. S. Kantor, and R. A. Flavell, Protection of Mice Against the Lyme Disease Agent by Immunization with Recombinant OspA, Science. 250:553-556, 1990, and sera containing antibodies against OspA has been shown to exhibit borreliacidal activity independent of complement. Callister, S. M., R. F. Schell, K. L. Case, S. D. Lovrich, and S. P. Day, Characterization of the Borreliacidal Antibody Response to B. burgdorferi in Humans: A Serodiagnostic Test, J. Infect. Dis. 167:158-164, 1992. Similarly, a bactericidal antibody directed against OspB has also been reported. Sadziene, A., M. Jonsson, S. Bergstrom, R. K. Bright, R. C. Kennedy, and A. G. Barbour, A Bactericidal Antibody to B. burgdorferi is Directed Against a Variable Region of the OspB Protein, Infect. Immun. 62:2037-2045, 1994.
Despite the appearance of a humoral immune response to OspA and OspB during the course of infection and the proven effectiveness of antibodies against these proteins in killing B. burgdorferi and preventing infection, disease symptoms generally do not resolve without treatment. Vaccination of mice after infection with B. burgdorferi has been shown to partially clear spirochetes from the bloodstream and also to reduce the severity of disease symptoms, however, it did not eliminate them from other tissues, nor did it alter the course of joint and heart involvement. Fikrig, E., S. W. Barthold, and R. A. Flavell, OspA Vaccination of Mice with Established B. burgdorferi Infection Alters Disease but not Infection, Infect. Immun. 61:2553-2557, 1993. This implies that B. burgdorferi may be able to occupy immunologically priveledged sites where it is not accessible to antibodies or that it is able to evade immune response by altering its outer surface. OspA and OspB are the best characterized of the B. burgdorferi antigens and have been implicated as virulence factors. For example, it has been shown that a monoclonal antibody to OspA inhibits association of B. burgdorferi with human endothelial cells. Comstock, L. E., E. Fikrig, R. J. Shoberg, R. A. Flavell and D. Thomas, A Monoclonal antibody to OspA Inhibits Association of B. burgdorferi with Human Endothelial Cells, Infect. Immun. 61:423-431, and loss of a functional OspB protein results in a dramatic decrease in pathogenicity of B. burgdorferi. Schwan, T. G., W. Burgdorfer, and C. F. Garon, Changes in Infectivity and Plasmid Profile of the Lyme Disease Spirochete, B. burgdorferi, as a Result of In Vitro Cultivation, Infect. Immun., 56:1831-1836, 1988; and Sadziene, A., A. G. Barbour, P. A. Rosa, and D. D. Thomas, An OspB Mutant of B. burgdorferi has Reduced Invasiveness in Vitro and Reduced Infectivity in Vivo, Infect. Immun. 61:3590-596, 1993. Since the discovery of OspA and OspB, several other surface lipoproteins have been identified that are immunologically recognized and may also play an important role in disease pathogenicity. These have been named accordingly OspC,D,E, and F, and are being investigated as possible vaccine candidates.
The Borrelia genome is unique among the pathogenic spirochetes in that it contains both linear and circular plasmids accounting for approximately 150-kb. Barbour, A. G., Plasmid Analysis of B. burgdorferi, the Lyme Disease Agent, J. Clin. Microbiol. 26:475-478, 1988. Considering that the size of the genome in B. burgdorferi has been measured at approximately 950-kb, plasmid DNA accounts for a large fraction of the total DNA. Ferdows, M. S., and A. G. Barbour, Megabase-Sized Linear DNA in the Bacterium B. burgdorferi, the Lyme Disease Agent, Proc. Natl. Acad. Sci. 86:5969-5973, 1989. In Borrelia hermsii, a relapsing fever species, the genes for major outer membrane proteins are also arrayed on linear plasmids, Ferdows and Barbour, supra. This unique feature can result in the loss of plasmids during growth which is especially true during cultivation in growth media, Schwan et al. supra. One advantage a loss of plasmids may confer on the spirochete is the ability to evade the immune response. For example, antibodies directed against OspA will not affect a spirochete that has lost the plasmid encoding the OspA protein. A disadvantage is that the spirochete will suffer a decrease of virulence capabilities. All of the outer surface lipoproteins discovered thus far are located on plasmids. OspA and OspB are co-transcribed on a 49-kb linear plasmid, Bergstrom, S., V. G. bundoc, and A. G. Barbour, Molecular Analysis of Linear Plasmid-Encoded Major Surface Proteins, OspA and OspB, of Lyme Disease Spirochete B. burgdorferi, Mol. Microbiol. 3:479-486, 1989, OspD is located on a 38-kb plasmid, Norris, S. J., C. J. Carter, J. K. Howell, and A. G. Barbour, Low-Passage-Associated Proteins of B. burgdorferi B31: Characterization and Molecular Cloning of OspD, a Surface-Exposed, Plasmid-Encoded Lipoprotein, Infect. Immun. 60:4662-4672, 1992, OspC on a 26-kb plasmid, Fuchs, R., S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Molecular Analysis and Expression of a Borrelia Gene Encoding a 22 kDa Protein (pC) in Escherichia coli, Mol. Microbiol. 6:503-509, 1992, and OspE and OspF are co-transcribed on a 45-kb linear plasmis, Lam, T., T. K. Nguyen, R. R. Montgomery. F. S. Kantor, E. Fikrig, and R. A. Flavell, Outer Surface Proteins E and F of B. burgdoferi, The Agent of Lyme Disease, Infect. Immun. 62:290-298, 1994.
B. burgdorferi has a cell organization similar to that of other spirochetes. The bacterium has an inner or cytoplasmic membrane that is surrounded by periplasmic flagella. The flagella wraps around the cytoplasmic cylinder making the cell motile by a corkscrew motion. These structures are covered by a peptidoglycan cell wall and an outer membrane. Although it stains as a gram negative bacterium, it is different from other gram negative bacteria in that the peptidoglycan layer is attached to the cytoplasmic membrane instead of the outer membrane. As such, the outer membrane of B. burgdorferi is quite fragile, often releasing from the cell as small vacuoles or "blebs". Various attempts have been made to study the various components of the cell structure to better understand its mechanisms of pathogenesis. These methods include detergent treatment of cells to separate inner and outer membrane components. Brusca, J. S., A. W. McDowall, M. V. Norgard, and J. D. Radolf, Localization of Outer Surface Proteins A and B in Both the Outer Membrane and Intracellular Compartments of B. burgdorferi, J. Bacteriol. 173:8004-8008, 1994; freeze-fracture electron microscopy to visualize membrane and membrane bound proteins, Radolf, J. D., K. W. Bourell, D. R. Akins, J. S. Rusca, and M. V. Norgard, Analysis of B. burgdorferi Membrane Architecture by Freeze-Fracture Electron Microscopy, J. Bacteriol. 176:21-31, 1994, and isopycnic centrifugation to separate inner and outer membrane proteins without the use of detergents, Bledsoe, H. A., J. A. Carroll, T. R. Welchel, M. A. Farmer, D. W. Dorward, and F. C. Gherardini, Isolation and Partial Characterization of B. burgdorferi Inner and Outer Membranes by Using Isopycnic Centrifugation, J. Bacteriol. 176:7447-7455, 1994.
A recent report has shown that spirochetes can be observed in the midgut, hemolymph, and saliva of Lyme Disease ticks by direct immunofluorescence assays at various stages of the feeding process. Using this technique, transmission of B. burgdorferi from a tick to a host was shown to be multi-step process that probably results in delivery of the spirochetes into the host via the salivary glands. Ribiero, J. M. C., Mather, T. N., Piesman, J., and Spielman, A., Dissemination and Salivary of Lyme Disease Spirochetes in Vector Ticks (Acari:Ixodidae), J. Med. Entomol, 24:201-205, 1987.